The goal is to create site-specific recombinase (SSR)-expressing transgenic rats in an efficient and unbiased manner through a transposon-mediated in vivo gene trap. Rats are very relevant for modeling human biology and disease because rats, unlike mice, possess key biological similarities to humans. While in vitro gene trap mutagenesis is useful for gene discovery, a rapid and efficient in vivo method is preferable for two key reasons: 1) an in vivo gene trap will reveal an authentic spatio-temporal expression pattern, and 2) these genetic resources of can then be immediately employed for genome manipulations in the rat. Piecemeal promoter analysis in transgenic animals is clearly not sufficient for rapidly and economically generating effective tools for SSR-mediated tissue-specific genome manipulation. The generation of transgenic rats is simply too expensive, and is performed at only a few institutions. Mouse promoter elements could be used to generate SSR-expressing transgenic rats, but most promoter elements lack key regulatory sequences, are sensitive to position effects at genomic insertion sites, or will not necessarily produce identical expression patterns across species. Considering that tens of thousands of transcribed elements likely exist in the mammalian genome, an efficient in vivo method is needed to authentically and rapidly recapitulate endogenous expression patterns to generate a diverse and effective set of transgenic tools. Furthermore, a method to identify new transcribed elements would accelerate a comprehensive understanding of mammalian genomics. Gene trap mutagenesis is a standard approach for identifying and exploiting new transcription units, but traditional strategies, such as those targeting embryonic stem cells, are simply untenable for a rapid and efficient in vivo screen. By using a PiggyBac (PB) transposon system that we have enhanced at Transposagen, we will pursue an in vivo promoter trap in rats. Promoter traps will be visualized in live embryos and animals using bright fluorescent proteins (FPs). The primary reporter in this promoter trap is a Cre-EGFP fusion protein, in which both Cre recombinase activity and EGFP fluorescence marks the expression domain of each trapped element. The Cre-EGFP cassette trap is delivered by a PB transposon that originates as a transgene concatemer in one transgenic line, called the "donor." Another transgenic line, the "driver," provides expression of the PB transposase in the germ line. Transposon mobilization is simply initiated by interbreeding driver and donor lines, such that the PB transposase mobilizes the Cre-EGFP gene trap transposon within germ cells in double transgenic animals, designated as "seed" rats. PB is the most efficient transposon yet described for gene mutagenesis in mammalian cells, and by using an enhanced PB transposase, we expect an insertion rate that should yield at least one gene trap event per gamete. As a result, each G1 offspring bred from seed rats will display a unique expression pattern of the Cre-EGFP reporter. Because it is an insertional mutagen, the integration site of the PB transposon is easily determined by simple PCR cloning techniques. EGFP fluorescence will be documented in E13.5 and E16.5 embryos and the identification of insertion sites will enable one to link a specific expression pattern with specific genomic locations. As noted above, tools are needed for expanding the genetic resources of the rat. A strategy to accelerate the tools for genetic manipulations in the rat is integral to our approach here. By using a Cre-EGFP reporter, we will be creating recombinase-mediated tools for conditional mutagenesis. To monitor Cre activity we will create transgenic rats that express a tdTomato fluorescent protein in cells following a Cre recombination event. The tdTomato protein exhibits very bright fluorescence, and has proven useful for in vivo expression analysis. Using the rat ROSA26 promoter that drives ubiquitous expression, tdTomato expression will be activated only following Cre-mediated removal of an intervening sequence. These transgenic rats will be mated to the G1 offspring described above that are obtained from seed rats. We will analyze live G2 embryos for EGFP and tdTomato fluorescence. By using these FPs, each gene trap can be monitored in real-time;this will create the opportunity for a further in depth analyses in future investigations. We will identify at least 25 unique Cre- expressing elements in this screen in live embryos, as a proof of concept. In addition to tdTomato, Cre recombinase activity will also trigger FlpERT2 expression, which is linked to the tdTomato open reading frame via an internal ribosomal expression sequence. FlpERT2 activity is dependent on 4-hydroxytamoxifen (4-OHT), and thus provides inducible Flp recombinase activity. Our design enables two key Flp-mediated manipulations: 1) Flp recombinase induction (via 4-OHT) removes the Cre-EGFP gene trap through flanking Flp recombinase target sites and 2) FlpERT2 expression will enable Flp-dependent conditional modification of FRT-containing alleles (generated through other efforts). The tools and resources generated here will provide great advances for rat genetics. This screen will identify at least 25 transcribed elements exhibiting a unique in vivo expression pattern of Cre-EGFP. Our strategy will provide the opportunity to easily generate hundreds of additional gene trap lines. Our investigation will generate valuable rat transgenic lines for recombinase-mediated conditional mutagenesis in future studies.